For MITF protein levels, cells were lysed after 48 hours of culture using RIPA-Buffer (50 mM Tris/Cl, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.25% Na-DOC, 0.1% Protease Inhibitor, pH 7.4). Protein concentration was quantified by Bradford protein assay (Protein Assay Kit, BioRad, Hercules, CA). The lysates were run on a 10% polyacrylamide gel. MITF was detected using anti-MITF (clone D5, ThermoFisher Scientific, 1:1000) and secondary mouse monoclonal IgG HRP-conjugated antibody (clone HAF007, R&D Systems, 1:2000). β-Actin mouse monoclonal IgG HRP-conjugated antibody (clone C4, Santa Cruz Biotechnology, Santa Cruz, CA; 1:3000) was used as a loading control. Chemiluminescence was measured using Thermo Scientific Super Signal West Pico PLUS Chemiluminescent Substrate (ThermoFisher Scientific, Waltham, MA) with ChemiDoc Imagining Systems (BioRad). Densitometry was performed with Image Lab Software (BioRad).
Thermo scientific supersignal west pico plus chemiluminescent substrate
Manufactured by Thermo Fisher Scientific
Sourced in United States, China
The Thermo Scientific™ SuperSignal™ West Pico PLUS Chemiluminescent Substrate is a laboratory reagent designed for the detection of protein and nucleic acid targets in various analytical techniques, such as Western blotting and immunoassays. The substrate generates a luminescent signal in the presence of the target analyte, which can be measured using a compatible detection system.
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Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Image lab software, by Bio-Rad (1 mentions)
Protein assay kit, by Bio-Rad (1 mentions)
P egfr, by Cell Signaling Technology (1 mentions)
Anti β actin antibody, by Merck Group (1 mentions)
Invitrogen ibright imaging system, by Thermo Fisher Scientific (1 mentions)
P smad2 smad3, by Cell Signaling Technology (1 mentions)
P erk antibody, by Santa Cruz Biotechnology (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Thermo scientific supersignal west femto chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Gel doc xr gel documentation system, by Bio-Rad (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
4 protocols using thermo scientific supersignal west pico plus chemiluminescent substrate
1
MITF Protein Expression Analysis
2
Western Blot Analysis of Corneal Epithelial Cells
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The CECs scraped off the corneas that served as controls during wounding and at the end of experiment were collected and frozen immediately in liquid nitrogen and stored at an Eppendorf tube at −80°C. For western blot, human and mouse CECs were lysed with radioimmunoprecipitation assay buffer. The lysates were centrifuged to obtain the supernatant. Protein concentration was determined by bicinchoninic acid (BCA) assay. The protein samples were separated by SDS-PAGE and electrically transferred onto nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were blocked with 3% BSA and subsequently incubated with primary and secondary antibodies. Signals were visualized using Thermo Scientific SuperSignal West Pico PLUS Chemiluminescent Substrate (#34580; Thermo Fisher Scientific, Waltham, MA, USA) using an Invitrogen iBright Imaging System (Thermo Fisher Scientific). Antibodies to phospho-Akt (p-Akt, #9271, 1:500 dilution), p-EGFR (#4470, 1:500 dilution), and p-SMAD2/SMAD3 (#8828, 1:500 dilution) were obtained from Cell Signaling Technology (Danvers, MA, USA); p-ERK antibody (#sc-7383, 1:1000 dilution) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA); and anti-β-actin antibody (#A1978, 1:10000 dilution), which served as the loading control, was obtained from Sigma-Aldrich (St. Louis, MO, USA.
3
CFTR Protein Detection Assay
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CFTR: FRT and 16HBEge cells were washed with PBS, harvested, and lysed with ice-cold RIPA buffer containing 1% EDTA and 1% protease inhibitors (Halt Thermoscientific protease inhibitor cocktail, Catalog # PI78425, Thermo Fisher). Protein concentrations were determined using the PierceTM BCA Protein Assay Kit (Catalog # PI23250, Thermo Fisher). Monoclonal CFTR primary antibody (UNC 596; 1:5000; received under MTA from the University of North Carolina at Chapel Hill) and mouse monoclonal anti-α-tubulin (1:5000; Catalog # 62204, Thermo Fisher) antibodies were used to detect CFTR and α-tubulin levels, respectively. Protein bands were visualized using Thermo Scientific™ SuperSignal™ West Femto Chemiluminescent Substrate (Catalog # PI34096, Thermo Fisher) for CFTR proteins or Thermo Scientific™ SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Catalog # PI34579, Thermo Fisher) for α-tubulin. Images were captured by a Gel Doc XR + Gel Documentation System (Bio-Rad).
4
Whole-Cell Lysates Protein Extraction and Analysis
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To collect whole-cell lysates, cells were harvested, washed with PBS, resuspended in pre-chilled RIPA lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, EDTA, protease inhibitors, and phosphatase inhibitors), and incubated on ice for 30 min. Lysates were supplemented with 4× loading buffer and boiled for 10 min. The protein concentration was determined using a BCA protein assay kit (Beyotime, Shanghai, China, P0010). Proteins in whole-cell lysates were separated on 8% or 10% SDS-polyacrylamide gels and transferred onto polyvinylidene difluoride membranes. Membranes were blocked with 1% bovine serum albumin at RT for 1 h, incubated with primary antibodies at 4°C overnight, and then incubated with secondary antibodies at RT for 1 h. Immunoreactive bands were detected using Thermo Scientific™ SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific, 34580) and a ChemiDoc™ MP Imaging System (Bio-Rad, Shanghai, China).
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